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A phase l study using intravesical Ad-IFNαSyn3 for patients with bacillus Calmette-Guerin-resistant superficial bladder cancer showed a complete remission (CR) of 43% at 90 days after treatment with high levels of interferon-α (IFNα) being produced. Ad-IFNα kills bladder cancer cells by two apoptotic and one necrotic mechanism that can be measured by soluble forms of cytokeratin 18 (CK18) using M30 and M65 ELISAs, assays for caspase-cleaved (apoptotic) and uncleaved (necrotic) cell death, respectively. Therefore, we determined whether M30 and M65 levels in the urine after treatment could document all three mechanisms of cancer cell kill and also predict having a CR. High levels of both M30 and M65 were found in all patients within 24 h after treatment with all three types of cancer cell death occurring. Moreover, the return of both M30 and M65 levels in the urine to normal levels within 5 days or more after treatment was strongly associated with obtaining a CR (P=0.003). This is the first time that such assays have been used to study response to therapy in the urine of patients with bladder cancer and in the future may prove valuable in predicting clinical outcome.

Over the past several years we have obtained considerable evidence indicating that adenoviruses-expressing interferon α (Ad-IFNα) can overcome resistance to the IFNα protein itself. Since cancer cells infected with Ad-IFNα also show high perinuclear cytoplasmic IFNα expression, we were interested in whether endoplasmic reticulum (ER) stress and cleavage of caspase 4 could have a major role in Ad-IFNα-produced cancer cell death. Indeed, procaspase 4 was upregulated and cleaved as early as 12 h after Ad-IFNα infection of the cancer cells, which co-localized with IFNα staining and ER tracker. In contrast, immortalized normal human urothelial cells, although exhibiting similar perinuclear IFNα staining, showed no cleaved caspase 4. Caspase 4 cleavage was not blocked by the caspase 8 specific inhibitor zIETD, indicating that caspase 4 activation was independent of caspase 8 activation. Blocking caspase 4 also inhibited activation of caspase 3 in Ad-IFNα containing cells. Finally, the cleaved form of caspase 4 (p10) was detected in Ad-IFNα-positive cancer cells from the urine of a patient following intravesical Ad-IFNα/Syn3 treatment. Therefore, ER stress and activation of caspase 4 appears to be an important mechanism involved in the direct cancer cell death produced by Ad-IFNα and also occurs in the clinical setting.